Mesenchymal stem cells are undifferentiated cells that exist in the mesenchymal tissues, and are known to have self-proliferation ability and differentiation ability into mesodermal cells such as osteocytes, adipocytes, and chondrocytes. Furthermore, it has been reported that mesenchymal stem cells have multipotency because they may be differentiated into non-mesodermal cells such as hepatocytes or myocardium where the heart is pulsating.
Thus, mesenchymal stem cells are expected to be used in the regenerative medicine, and clinical studies are being performed on cell therapy to transplant mesenchymal stem cells into sites where the self-renewal may not be easily achieved.
In order to differentiate mesenchymal stem cells into specific tissue cells, factors for inducing the differentiation of cells are needed. Currently, studies on specifying factors which may induce the differentation of mesenchymal cells into bones, fat, cartilage, liver and myocardium are being performed, and the differentiation into each tissue has been efficiently induced.
The induction of the differentiation of mesenchymal stem calls is achieved by ding cells onto a petri dish and culturing them under two-dimensional conditions.
However, when the differentiation of mesenchymal stem cells is induced under this two-dimensional environment, properties of the cells become different from original properties of in vivo tissues which have a three-dimensional structure. In particular, when the induction of the differentiation of mesenchymal stem cells into cartilage is performed in a pad dish, most of the mesenchymal stem cells are known not to be differentiated into chondrocytes.
Therefore, when mesenchymal stem cells induced to differentiate into chondrocytes, the differentiation of mesenchymal stem cells in which three-dimensional aggregates are formed must be induced.
However, mesenchymal stem cells are proliferated flatways and singularly only on pew dishes, and the self-aggregating properties are not exhibited as in hepatocytes.
Therefore, methods, such as a pellet culture method, are usually used as a forted method for aggregating mesenchymal stem cells (Refer to Non-Patent Document 1).
In addition, a culture method using a three-dimensionally shaped culture support having a plurality of concave portions is also considered as described in Patent Document 1.
However, a pellet culture method described in Non-Patent Document 1 is a method for aggregating cells by adding a suspension of the cells to a 15 ml centrifuge tube and precipitating the cells forcibly by centrifugation, and it has been difficult to obtain good cell aggregates because mechanical stimuli on cells are strong so that the cells are damaged.
In addition, when aggregated mesenchymal stem cells are induced to differentiate into chondrocytes by a pellet culture method, expression of Type II collagen or aggrecan which is a gene expressed by in vivo chondrocytes (hyaline chondrocytes) may be identified.
However, the expression of Type X collagen which is exp by further differentiating hypertrophic chondrocytesor CD105 specific to mesenchymal stem cells may be also identified and thus there is a problem in that the aggregate cannot be induced to differentiate into uniform cartilage tissues having the same properties in an in vivo tissue.
In addition, when a three-dimensionally shaped culture support having a plurality of concave portions as described in Patent Document 1 is used there is a problem in that mesenchymal stem cells cannot be aggregated only into concave portions and thus the same properties cannot be obtained in an in vivo tissue, and the induction of differentiation into a tissue having more uniform differentiation conditions cannot be achieved.
Thereto there is a need for a cell culture support and a cell culture method in which a cell aggregate can be formed by stationary culture of mesenchymal stem cells to thereby be induced to differentiate into a tissue which has the same properties and has uniform differentiation conditions in in vivo tissues.
[Patent Document 1] JP-A-2008-306987
[Non-Patent Document 1] Mark F, Pitternger et al., Science, 284, 1999, p. 143-146